Single-molecule fluorescence spectroscopy was used to study the behavior of a 2-hydroxy substituted nile red (NR-OH) dye in silica films incorporating amine gradients. These amine gradients were made by the vapor-phase deposition of 3-aminopropyltrimethoxysilane (3-APTMOS) onto microporous silica base layers in a humidity-controlled chamber. Water contact angle and ellipsometry measurements were used to confirm the formation of amine gradients. X-ray photoelectron spectroscopy (XPS) measurement of the nitrogen content of the gradient films showed that there was a gradual decrease in the amine content of the gradient films away from the 3-APTMOS reservoir. Bulk fluorescence studies of the dye in basic media of varying strengths showed that there is a hypsochromic shift in the emission maxima of the spectra with increasing basicity. Two-color fluorescence videos acquired along the gradients showed that the fluorescence of the dye was more intense in the 580/40 nm channel (where the deprotonated form of the dye is more prevalent) than the 640/40 nm channel at the high amine end of the films. With decrease in the amine content of the films, the fluorescence of the 580/40 nm channel decreases relative to the 640/40 nm channel. This study can aid in understanding the nature of active sites of bifunctional catalysts for an aldol condensation reaction carried out at the single molecule level.